A study of the dynamics of the protein core of the L99A mutant of T4 lysozyme using nuclear magnetic resonance relaxation dispersion

Many biologically important functions of proteins occur in the ms-μs time scale. The L99A cavity mutation of T4 lysozyme provides an ideal system to study motional dynamics at this time scale. The L99A mutation generates a 150 A3 cavity inside the protein core. The cavity can bind many different kinds of non-polar molecules and the lifetime of the bound states of these ligands is in ms-μs regime. In collaboration with Lewis Kay's group, we developed and applied the relaxation dispersion NMR methods to the study these motions around the cavity in more detail. The exchange rate constants, equilibrium constant, and chemical shift changes of the “cavity closed” and “cavity open” states were obtained. These measurements provide us insight into the possible ligand binding pathway and a direct way to monitor the ms-μs dynamics. It is shown that most of the methyl groups in the protein are not involved in any two-state exchange process, but all those methyl groups that are active in the two-state exchange process are located in or around helix F, G, I and J. This region appears to form a gateway between the cavity and the bulk solvent. By studying the slow motion at different temperatures, we showed that the free energy difference between the cavity closed state and the cavity open state was lowered by increasing the entropy of the cavity open state as compared to the cavity close state. The NMR relaxation dispersion approaches described in this dissertation will be of great use for the study of ms-μs dynamics in many protein systems.